The present invention generally relates to sizing of particles, for example biological cells and related biological particles such as nuclei, chromosomes and other sub-cellular organelles, through analysis of signal pulses from either fluorescence or light scatter in flow cytometers or similar flow meters, and, more particularly, relates to methods and apparatus for differentiating particles of different size, shape or orientation from one another.
FLow cytometers and similar devices for measuring particle characteristics, not limited to biological particles, generally operate by conveying particles one-by-one through a beam of exciting electromagnetic radiation, for example from a laser, and responding to radiation resulting from interaction of the particle with the beam to output a signal in the form of a pulse as the particle enters and passes through the beam. Two examples of radiation resulting from interaction of the particle with the beam are scattered light and secondary fluorescence.
There is presently an interest in the accurate sizing of live biological cells both for clinically useful applications, and for the answering of important questions in cell biology. Accordingly, a number of approaches have been proposed for determining cell size non-destructively employing flow cytometers.
One approach to determining cell size has been to use intensities of light scattered at small angles. The intensity of light scattered in the near forward direction is predominantly due to diffraction (first ring of the diffraction pattern) and is roughly proportional to the cross-sectional area of the cell. There are, however, a number of problems with this light scatter approach. For one, the angular width of the first ring of the diffraction pattern decreases with increasing cell size. Another problem is that the use of laser beam dumps or obscuration bars in commercial flow cytometers modifies the proportion of the scattered light which reaches the detectors, thereby leading to a non-systematic relationship between scattered light intensity and cell cross-sectional area. A third problem is that at angles greater than those subtended by the main diffraction ring from the cell, typically 2.degree. to 3.degree. for most mammalian cells, the scattered light intensity is strongly dependent on cell refractive index. Two cells of the same size but of different refractive index, sometimes reflecting changes in internal cell structure, will have different light scattering intensities. Light scatter intensity in the above-described approach is thus not always a reliable measure of cell size.
A different approach to sizing of live biological cells, and more particularly to determining nuclear to cytoplasmic (N/C) ratio, is a "slit-scan" or "programmed pulse shape analysis" technique whereby the fluorescence pulse shapes of Acridine Orange metachromatically stained cells passing through a slit focused laser beam (typically five micrometers in width) are subsequently analyzed by a level detection algorithm which defines the nuclear boundary, thereby enabling nuclear-to-cell size measurements to be determined on a cell-by-cell basis. This technique is described in the Wheeless, Jr., et al U.S. Pat. No. 3,657,537, and in the following two literature references: L. L. Wheeless, Jr. and S. F. Patten, Jr., "Slit-Scan Cytofluorometry", Acta Cytol., Vol. 17, No. 4, pp. 333-339 (1973); and L. L. Wheeless, Jr., A. Hardy and N. Balasubramanian, "Slit-Scan Flow System for Automated Cytopathology", Acta Cytol., Vol. 19, No. 1, pp. 45-52 (1975).
Another sizing technique is described in the Coulter U.S. Pat. No. 3,961,249 which discloses a particle size analyzer responding to output pulses from apparatuus such as is disclosed in the Coulter U.S. Pat. No. 2,656,508 utilizing what is known as the Coulter principle. As is pointed out in the Coulter U.S. Pat. No. 3,961,249, the amplitude of a Coulter pulse is an indicator of cell or particle size. It is pointed out in this Coulter U.S. Pat. No. 3,961,249 that another indication of particle size may be obtained by differentiating one of the edges of the Coulter counter particle pulse, preferably the trailing edge. Specifically, the peak value of the derivative is an indicator of particle size.
Another prior art sizing technique may be generally termed "time-of-flight" wherein output pulse width of a cytometer is measured, with the pulse resulting either from fluorescence or light scatter. Various specific time-of-flight measurement techniques are described in the literature reference: T. K. Sharpless and M. R. Melamed, "Estimation of Cell Size From Pulse Shape in FLow Cytofluorometry", J. Histochem. Cytochem., Vol. 24, No. 1, pp. 257-264 (1976). It should be pointed out that the present invention is specifically directed to new apparatus and new methods for analyzing previously known signal pulses similar to those pulses described in the above cited Sharpless and Melamed article. Accordingly, to the extent that the Sharpless and Melamed article is useful for understanding the nature of cytometer pulses and the manner in which they are obtained, the entire disclosure of the above cited Sharpless and Melamed article entitled "Estimation of Cell Size From Pulse Shape in Flow Cytofluorometry" is hereby expressly incorporated by reference. However, the particular methods and apparatus by which these pulses are analyzed according to the present invention to yield size information are not disclosed in the Sharpless and Melamed article, and there are corresponding details of these signal pulses which, although present in the actual pulses, are not described in the Sharpless and Melamed article.
As described in the Sharpless and Melamed article, cytometer output pulses can be analyzed in various ways to provide particle size estimates.
As Sharpless and Melamed point out, the most straightforward approach would be to measure overall pulse width employing a threshold level set just high enough to exclude background noise. Measured pulse width then corresponds to that portion of the pulse which begins where pulse amplitude crosses the threshold level on the rising edge of the pulse, and ends where pulse amplitude re-crosses the threshold level on the falling edge of the pulse. One drawback to this is that the measurement is made in a region of intrinsically low signal-to-noise ratio. A more serious problem, however, particularly where fluorescence is measured to produce the output pulses, is that fluorescence staining of cells is inherently non-uniform such that two cells of the same size may have different fluorescence intensities, and the result from the fixed threshold pulse width measurement technique depends upon absolute pulse amplitude, as well as on pulse shape. Accordingly, even if two cells have the same size, the bright one will incorrectly be measured as larger. Fluorescence staining is non-uniform due not only to processing variations as a practical matter, but also due to life cycle changes in biological cells which affect the degree of stain absorption.
To circumvent this, Sharpless and Melamed propose a number of amplitude independent estimators for analyzing cytometer pulses to determine particle width. A first evaluation method is a peak width (PW) method wherein total pulse width is measured against a threshold level scaled to some fraction of the pulse height. This result is said to depend only on pulse shape. A second method is a quantile width (QW) method wherein the time required to accumulate some fixed central fraction of the total intensity is recorded by a pair of thresholds scaled to the final value of the integral. Both the peak width (PW) and the quantile width (QW) method require that the pulse shape be stored in a high quality delay line until the peak height or total intensity has been measured and held. A third amplitude independent measurement is the ratio of pulse area to peak height, which is abbreviated "AW".
The approaches described by Sharpless and Melamed generally require the laser beam to be small compared to the cell size. In particular, the effective aperture through which cells or other particles pass is established by means of a slit focused laser beam having a Gaussian intensity distribution, as this is the approximate beam intensity distribution found on most commercially available cytometers. The beam width, and therefore effective aperture, employed ideally is as small as possible, and typically is in the order of or slightly less than the width of the cells being measured. The narrower the beam, the more accurately the pulse shape represents the actual particle shape, and the less it represents the beam intensity distribution. The available beam widths, however, are generally not as narrow as in the Wheeless et al slit-scan systems.
Yet another approach to measuring dimensional characteristics of particles is disclosed in the Curby U.S. Pat. No. 3,919,050. Curby describes a method for analyzing the shape of pulses from either a Coulter Counter or an optical sensor to provide information characterizing the particles producing the pulses. In the Curby method, a secondary pulse producing means is triggered at particular times during the duration of each of the particle pulses to produce secondary voltage pulses whose amplitudes reflect the amplitude of the particle pulse at the time of triggering. These secondary voltage pulses reflect the shape of the primary pulse, and are used to characterize the particles being analyzed.